GSM7838556: RT7 4-OHT TAF10 rep2; Mus musculus; OTHER
Cells were washed and resuspended in wash buffer and incubated for 10 min at room temperature with 10 µl of concanavalin A-coated beads (Bangs Laboratories, BP531). Cells bound to beads were permeabilized using 0.05% digitonin and incubated with the appropriate antibody at 4°C overnight. Protein A-MNAse (pA-MN) was added to a final concentration of 700 ng/ml and incubated at 4°C for 1 hr on a tube rotator. After washing, pA-MN was activated with 2 µl of 100 mM CaCl2 and digestion was performed for 30 min at 0°C. The reaction was stopped with 100 µl of stop buffer containing 2 pg/ml of heterologous spike-in DNA from yeast. Release of the DNA fragments was achieved by incubating samples at 37°C during 15 min. DNA was extracted using NucleoSpin columns (Macherey-Nagel) and eluted in 30 µl of NE buffer. CUT&RUN-seq libraries were generated using the MicroPlex library preparation kit v3 (Diagenode), following the manufacturer's instructions, except that the stage 4 of the library amplification PCR was performed with a combined annealing-extension step for 10 s at 60°C, and stage 5 with an extension step for 10 s at 60°C for 7 cycles. Yield and size distribution were quantified on a 2100 Bioanalyzer instrument (Agilent).